title: Agrobacterium tumefaciens-mediated transformation of the dermatophyte, Trichophyton mentagrophytes: an efficient tool for gene transfer.
abstract: Agrobacterium tumefaciens-mediated transformation (ATMT) was used to facilitate gene transfer into the clinically important dermatophyte, Trichophyton mentagrophytes (teleomorph: Arthroderma vanbreuseghemii). A binary vector containing a hygromycin B resistance cassette was introduced into A. tumefaciens, and the resultant strain was co-cultivated with fungal small conidia. Transformation yielded a large number of hygromycin B-resistant transformants. Hybridization analysis showed that most of the transformants harboured a single copy of T-DNA randomly integrated into the genome. Transformation frequency was increased to more than 200 per 10(7) conidia by optimizing the co-cultivation time and temperature. ATMT was then used for targeted gene disruption mediated by homologous recombination. Using a PCR-based strategy, we isolated the areA/nit-2-like nitrogen regulatory gene (tnr:Trichophytonnitrogen regulator) from T. mentagrophytes. A binary vector containing two regions of the tnr locus flanking the hygromycin B resistance cassette was constructed and introduced into T. mentagrophytesvia ATMT. Transformants with disruption of the areA/nit-2-like gene (tnr) were obtained in three of four independent disruption experiments, most of which showed homologous recombination via double crossover without additional ectopic integration of the disruption construct.
authors.affiliation: Teikyo University Institute of Medical Mycology, Teikyo University, Hachioji, Tokyo, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yamada
authors.forename: Koichi
authors.initials: K
authors.lastname: Makimura
authors.forename: Kazuo
authors.initials: K
authors.lastname: Satoh
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Umeda
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Ishihara
authors.forename: Shigeru
authors.initials: S
authors.lastname: Abe
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D003883:Arthrodermataceae / Q000235:genetics / Q000254:growth & development; D018076:DNA, Complementary / Q000235:genetics; D018014:Gene Transfer Techniques; D005800:Genes, Fungal; D010957:Plasmids / Q000235:genetics; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics*; D014249:Trichophyton / Q000235:genetics* / Q000254:growth & development
chemical_list: D018076:DNA, Complementary; D014157:Transcription Factors